Oxygenase enzymes must coordinate the delivery of four protons and four electrons to O2 in order to prevent the formation of harmful, partially reduced reactive oxygen species. The risks posed by reactive intermediates are so great that aerobic organisms need mechanisms to protect oxygen-utilizing enzymes from inactivation when primary electron/proton transfer mechanisms are disrupted. Radical transfer pathways that deliver strongly oxidizing holes from frustrated reactive intermediates in enzyme active sites to the protein surface for reaction with intracellular antioxidants can provide such protection. These radical transfer pathways are likely constructed from chains of Trp, Tyr, Cys, and possibly Met residues. This research program will focus on members from two superfamilies of this enzyme class: the cytochromes P450 (P450) and the 2- oxo-glutarate dependent nonheme iron oxygenases (2OG-Fe). The cytochromes P450 are members of a superfamily of heme oxygenases that perform xenobiotic metabolism and biosynthesis. In mammals these functions include drug metabolism, conversion of lipophilic molecules to more polar products for enhanced elimination, steroid biosynthesis, and eicosanoid synthesis and degradation. Cytochromes P450 also are responsible for 66% of enzymatic activation of carcinogens. Elucidating the mechanisms by which P450s avoid inactivation in the presence of diverse substrates can contribute to defining therapeutic drug efficacies and mitigating the risks of adverse drug-drug interactions. Drugs for cancer treatment and prevention have been designed to target P450s through competitive inhibition and mechanism based irreversible inhibition. To understand the biological response of P450s to these compounds, it is essential to delineate the mechanisms that the enzymes use to protect themselves against degradation. Enzymes from the 2OG-Fe superfamily use 2-oxoglutarate as a 2-electron donating co-substrate, Fe2+ as a cofactor, and O2 to effect the hydroxylation of organic substrates. The 60-70 human 2OG-Fe enzymes exhibit a wide array of biological functions including collagen biosynthesis, lysyl hydroxylation of RNA splicing proteins, DNA repair, RNA modification, chromatin regulation, epidermal growth factor-like domain modification, hypoxia sensing, and fatty acid metabolism. The roles of distance, driving force, and proton acceptors will be probed in systematic investigations of Tyr, Trp, Cys, and Met radical generation reactions in a robust cupredoxin (Aim 1). Potential functional radical transfer pathways have been identified in two cytochromes P450 (Aim 2) and two 2OG-Fe enzymes (Aim 3). Catalytic turnover, enzyme deactivation kinetics, and photochemical electron-transfer measurements will be performed to evaluate the enzyme protective capacities of these charge transport pathways.